The m6A reader MhYTP2 regulates MdMLO19 mRNA stability and antioxidant genes translation efficiency conferring powdery mildew resistance in apple

Summary N6‐methyladenosine (m6A) reader protein plays an important role in trichome morphology, developmental timing and morphogenesis in Arabidopsis. However, the function of m6A readers in plant‐microbe interaction remains unclear. Here, a Malus YTH‐domain family protein MhYTP2 was initially characterized as an m6A reader. MhYTP2 overexpression increased mRNA m6A modification level and translation efficiency. The m6A in the exon regions appeared to destabilize the mRNAs, whereas m6A in the untranslated regions positively correlated with the associated mRNA abundance. MhYTP2 overexpression enhanced apple powdery mildew resistance, possibly by rapidly degrading the bound mRNAs of MdMLO19 and MdMLO19‐X1 and improving the translation efficiency of the antioxidant genes. To conclude, the results shed light on the apple m6A profile, the effect of MhYTP2 on m6A profile, and the m6A roles in MdMLO19 and MdMLO19‐X1 mRNAs stability and glutamate dehydrogenase 1‐like MdGDH1L mRNA translation efficiency.


Introduction
Biotic stresses, including fungal and bacterial diseases, inevitably challenge plant survival and reproduction. Powdery mildew (PM) caused by Podosphaera leucotricha is a devastating disease of apple (Malus × domestica Borkh.), one of the most widely cultivated and economically important fruits worldwide. PM causes white spots on young green tissues, and the infected leaves crinkle, curl and prematurely drop. Blossoms and fruits are not the primary targets of the PM fungi; however, infection of these tissues is also possible (Foulongne et al., 2003;Turechek and Carroll, 2004;Xiao et al., 2001). Subsequently, PM affects apple fruit development and quality, reducing fruit yield and market value.
Plants have evolved several defence mechanisms over time. The plant disease stress responses involve molecular, physiological and cellular adaptations. At the molecular level, plants control gene expression levels by reprogramming their genetic machinery, via which they implement defence mechanisms and increase stress tolerance to minimize the effects of the accompanying biological damage (Fujita et al., 2006). Genetic regulatory networks, including post-transcriptional control of gene expression, serve as powerful strategies in disease stress responses (Huh and Paek, 2014). For example, apple Mildew Locus O 19 (MdMLO19) plays a pivotal role in PM resistance; its knockdown significantly induced PM resistance (Pessina et al., 2014(Pessina et al., , 2016. Meanwhile, the absence of Mlo primes the responsiveness for the onset of multiple defence functions in barley (Büschges et al., 1997;Wolter et al., 1993). RNA binding proteins (RBPs), the important components of such regulatory networks that remain active during and after transcription (Chen and Varani, 2013), identify and combine with target RNAs via the RNA binding domains (RBDs). Among the various post-transcriptional modifications, N 6methyladenosine (m 6 A) RNA methylation is one of the most pivotal internal modifications and a conserved post-transcriptional mechanism that enriches and regulates genetic information in eukaryotes (Yue et al., 2019). The m 6 A modification accounts for 80% of the RNA base modifications in eukaryotic cells (Kierzek and Kierzek, 2003). Studies have shown that m 6 A modification in plants influences various growth and development phenomena, including embryo development and seed germination (Vespa et al., 2004;Zhong et al., 2008), apical meristem and flower development (Bodi et al., 2012;Duan et al., 2017;Růžička et al., 2017;Shen et al., 2016), microspore development (Zhang et al., 2019), root development (Chen et al., 2018), leaf surface trichome development (Arribas-Hernández et al., 2018;Bodi et al., 2012;Chen et al., 2018;Scutenaire et al., 2018;Vespa et al., 2004;Wei et al., 2018), and fruit ripening (Zhou and Tian, 2019).
Plant m 6 A modification influences stress responses also. Studies have shown that the Arabidopsis m 6 A reader proteins evolutionarily conserved C-terminal region1 (ECT1) and evolutionarily conserved C-terminal region2 (ECT2) interact with stress response protein calcineurin B-like-interacting protein kinase1 (CIPK1) (Ok et al., 2005), and ECT2 affects mRNA relocation under heat stress (Scutenaire et al., 2018;Wei et al., 2018). Arabidopsis m 6 A demethylase ALKBH9B interacts with the viral coat protein and RNA and modulates alfalfa mosaic viral infection (Martí nez-Pérez et al., 2017). Meanwhile, the m 6 A hypomethylation plays a positive role in drought response in maize (Miao et al., 2020). Studies have demonstrated that these functions of m 6 A modification in stress responses were achieved through m 6 A regulating mRNA processing and metabolism, including mRNA transport, degradation (Luo et al., 2014;Meyer and Jaffrey, 2014;Schwartz et al., 2013;Shi and Wei, 2019), stability (Wang et al., 2014), splicing (Zhao et al., 2014) and translation efficiency Zhou et al., 2015). The role of methylation depends on the m 6 A reader protein (Bi et al., 2019;Shi et al., 2019). Arabidopsis ECT2 protein, containing an YTH domain, has been proven to be an m 6 A reader protein (Scutenaire et al., 2018;Wei et al., 2018). However, the role of apple RBP MhYTP2, a homologue of Arabidopsis ECT2, in the m 6 A binding function is unknown. Moreover, the m 6 A methylation machinery and the m 6 A characteristics and functions responsible for regulating pathological processes of horticultural crops remain largely unknown.
One of the major stress response mechanisms at the physiological level is the increase in the levels of antioxidants, such as ascorbic acid (AsA) and glutathione (GSH), to reduce stressinduced intracellular ROS accumulation (Gururani and Venkatesh, 2015). The AsA-GSH cycle, an antioxidant system, plays an important role in scavenging hydrogen peroxide (H 2 O 2 ) under stress (Wang et al., 2012). GSH, a major endogenous antioxidant, participates in H 2 O 2 detoxification via various glutathione peroxidases (Gu and Chauhan, 2015). Meanwhile, the GSH synthesis regulated by GDH helps plants absorb the excessive NH þ 4 to produce glutamate, for the first committed step in GSH synthesis (Skopelitis et al., 2006;Tercé-Laforgue et al., 2013;Yan et al., 2021). However, the direct role of GDH in PM resistance in apple is unclear.
Therefore, the present study aimed at characterizing the Malus YTH domain-containing RNA binding protein 2 (MhYTP2). The MhYTP2 was cloned from Malus hupehensis (pamp.) Rehd., a PM-resistant genotype, and overexpressed in Malus domestica cv. 'Roya Gala' to explore its function in PM resistance. Further, a global mRNA methylation and transcription analysis of the 35S:: MhYTP2 line OE-2 and the WT plants was performed to examine the influence of MhYTP2 on the overall methylation level. RNA immunoprecipitation (RIP)-sequencing was conducted to identify the targets of MhYTP2, and ribosome profiling (Ribo-seq) was performed to analyse the role of MhYTP2 in regulating translation efficiency. Collectively, our work demonstrates that the m 6 A binding function of MhYTP2 controls PM resistance by binding and affecting the mRNA stability of MdMLO19 and MdMLO19-X1 while facilitating the translation efficiency of antioxidant genes.

Results
Overexpression of MhYTP2 increases resistance to PM Three 35S::MhYTP2 lines (OE-1, OE-2, OE-3) showed increased resistance to PM compared with the WT plants in the pots under field conditions (Figure 1a). The 35S::MhYTP2 lines and the WT plants were also tested for their susceptibility to PM under laboratory conditions by artificial inoculation in the following study. Similarly, the 35S::MhYTP2 lines demonstrated lower disease severity than the WT plants 15 days after PM inoculation under laboratory conditions. Although the leaves of the 35S:: MhYTP2 lines were partially infected, the spread of spores on the adaxial leaf surface was significantly reduced compared with the WT (Figure 1b). The WGA staining revealed that the pathogen's spread in the 35S::MhYTP2 lines was significantly reduced compared with the WT (Figure 1c). Besides, the trypan blue staining indicated fewer disease symptoms in 35S::MhYTP2 lines than in the WT (Figure 1d).

Overexpression of MhYTP2 modifies the expression levels of m 6 A writers and erasers
Arabidopsis ECT2 protein has been reported as an m 6 A reader (Scutenaire et al., 2018;Wei et al., 2018). Sequence alignment revealed that MhYTP2 is a homolog of Arabidopsis ECT2 protein with a YTH domain ( Figure S1). Therefore, we hypothesized that MhYTP2 is also an m 6 A reader and may affect the mRNA methylation level. The expression levels of several important methyltransferase and demethylase genes (Wei et al., 2018) in the 35S::MhYTP2 lines and WT plants were analysed to investigate the effect of MhYTP2 on the mRNA methylation levels. The quantitative real-time PCR (RT-PCR) analysis revealed that the expression levels of the N 6 -adenosine-methyltransferase genes MT-A70-like (MdMTA) and non-catalytic subunit MTB (MdMTB) in the 35S::MhYTP2 lines were slightly lower than that in the WT plants. Meanwhile, the FKBP12-interacting protein of 37 kDa (MdFIP37) in the 35S::MhYTP2 lines was significantly lower than that in the WT plants ( Figure 2a). The transcript levels of demethylases MdALKBH2 and MdALKBH9B were similar in the 35S::MhYTP2 lines and WT plants; however, the transcript level of MdALKBH6 was significantly lower in the 35S::MhYTP2 lines than in the WT (Figure 2b). These observations indicate that MhYTP2 may influence the overall methylation level in apple.
Variations in methylome between 35S::MhYTP2 line OE-2 and WT Further, m 6 A-seq (Dominissini et al., 2013) was performed to assess the transcriptome-wide m 6 A methylation in the WT and 35S::MhYTP2 line OE-2 using leaf samples to investigate the potential influence of MhYTP2 on mRNA methylation. The mRNA samples from WT and 35S::MhYTP2 line OE-2 were fragmented into approximately 100 nucleotide-long oligonucleotides (input) before IP using an anti-m 6 A affinity purified antibody. Libraries were prepared from input control and IP fragments and subjected to parallel sequencing. The mRNA samples were prepared to perform two independent m 6 A-seq experiments. Highconfidence m 6 A peaks detected in both biological replicates for each line were used for subsequent analysis. A total of 15,225 and 17,303 high-confidence m 6 A peaks were identified from the WT and 35S::MhYTP2 line OE-2 leaves (fold change ≥ 2; P < 0.05), respectively. Here, 12,484 transcripts displayed increased m 6 A levels, while only 2024 transcripts showed decreased m 6 A enrichment in 35S::MhYTP2 line OE-2 compared with the WT plants (fold change ≥ 2; P < 0.05), suggesting a global increase in m 6 A methylation in 35S::MhYTP2 line OE-2 compared with the WT plants ( Figure 3a Figure 3b). Further, the distribution of m 6 A peaks in the whole apple transcriptome was analysed. Each transcript was divided into three non-overlapping segments based on the reference annotation: 5 0 untranslated region (UTR), coding sequence (CDS) and 3 0 UTR. As shown in Figure 3c, the meta-genomic profiles of m 6 A peaks in all samples indicated that m 6 A modifications were highly enriched around the CDSs and the 3 0 UTRs. Further, the distributions of m 6 A peaks in the WT and 35S::MhYTP2 line OE-2 plants were analysed. The percentage of m 6 A peaks in the CDS regions was lower in the 35S::MhYTP2 line OE-2 plants (48.30%) than that in the WT plants (53.20%), while those in the 5 0 UTRs (12.50% vs. 10.00%) and the 3 0 UTRs (39.20% vs. 36.80%) were higher. The m 6 A modifications around the UTRs in the 35S:: MhYTP2 line OE-2 were highly enriched than in the WT plants (Figure 3c), revealing the effect of MhYTP2 on the stability and translation of the m 6 A-modified mRNA (Luo et al., 2020;Wang et al., 2014).
Among the gene transcripts containing m 6 A modification, the majority contained one m 6 A peak (81.04%), followed by a few with two m 6 A peaks (16.27%); only a few exhibited three or more peaks (2.69%) (Figure 3d), with levels consistent with those in Arabidopsis and tomato (Luo et al., 2014;Wan et al., 2015;Zhou et al., 2019). Besides, hypergeometric optimization of motif enrichment (HOMER; http://homer.ucsd.edu/homer/) was applied (Heinz et al., 2010) to identify the sequence motifs enriched within the m 6 A peaks in apple. Clustering of the m 6 A peaks using  , and maize (Miao et al., 2020). These data suggest the conservation in m 6 A modification across plant species.
Studies have reported that m 6 A deposition influences mRNA abundance (Duan et al., 2017;Luo et al., 2014;Shen et al., 2016;Wan et al., 2015;Wang et al., 2014;Wei et al., 2018;Zhao and Roundtree, 2017). Therefore, RNA-seq was performed to evaluate the potential correlation between m 6 A mRNA methylation and gene transcript levels in apple. Comparison of the differentially expressed genes (DEGs; fold change ≥ 2; P < 0.05) identified via RNA-seq with the transcripts showing altered m 6 A levels between the 35S::MhYTP2 line OE-2 and WT plants revealed that the m 6 A modification in the exon regions appeared to destabilize the mRNAs, whereas m 6 A in the UTRs was positively correlated with the abundance of associated mRNAs ( Figure 3e). MdMLO19, a well-known S-gene involved in apple defence against PM (Pessina et al., 2014(Pessina et al., , 2016, was not identified in the 35S:: MhYTP2 line OE-2; however, the 3 0 UTR of MdMLO19 was m 6 Amodified in the WT plants. In addition, MdMLO19-X1 was identified in the 35S::MhYTP2 line OE-2 with m 6 A hypermethylation in the exon regions than the WT plants ( Figure 3f). MdMLO19-X1 is an isoform of the previously reported MdMLO19 (Pessina et al., 2014(Pessina et al., , 2016, with an mRNA sequence similarity of 52.96%, and an amino acid sequence similarity of 63.01% (Table S1). In fact, in addition to MdMLO19 and MdMLO19-X1, MdMLO1, MdMLO1-like and MLO11-like X2 that have not been tested for their function in PM resistance in apple were also found with variations in methylome between 35S::MhYTP2 line OE-2 and WT.
Furthermore, m 6 A methylation has been demonstrated to decrease mRNA stability (Duan et al., 2017;Shen et al., 2016;Wang et al., 2014;Zhao et al., 2017). Considering the role of m 6 A modification in exon regions in mRNA destabilization and that in UTRs in mRNA stability, we hypothesized that MhYTP2 promoted the degradation of MdMLO19 and MdMLO19-X1 transcripts. Therefore, the lifetime of MdMLO19 and MdMLO19-X1 transcripts was measured by blocking the transcription with actinomycin D. The MdMLO19 and MdMLO19-X1 transcripts were degraded rapidly after actinomycin D treatment in the 35S::MhYTP2 line OE-2 compared with the WT plants ( Figure 3g), which suggests that m 6 A modification promotes MdMLO19 and MdMLO19-X1 mRNAs degradation. These observations indicate that the site-specific m 6 A modification destabilizes MdMLO19 and MdMLO19-X1 mRNAs. Further quantitative RT-PCR analysis revealed significantly lower expression levels of MdMLO19 and MdMLO19-X1 in the 35S:: MhYTP2 line OE-2 than the WT plants after PM inoculation ( Figure 3h).

YTH-domain protein MhYTP2 is an m 6 A reader and
MhYTP2 associates with m 6 A-containing MdMLO19 and MdMLO19-X1 MhYTP2 was identified as a homolog of Arabidopsis ECT2 protein with a YTH domain ( Figure S1). Studies have proven ECT2 as an  m 6 A reader protein (Scutenaire et al., 2018;Wei et al., 2018). RIP sequencing was performed using 35S::MhYTP2-Flag apple calli line MhYTP2-F2 to determine whether MhYTP2 can bind with m 6 A-containing MdMLO19 and MdMLO19-X1 mRNAs (Figure S2). Unfortunately, MdMLO19 and MdMLO19-X1 mRNAs were not detected in MhYTP2-flag IP mRNA samples (Figure 4a). Consequently, the MhYTP2-His protein from Escherichia coli BL21 was expressed and purified to perform the in vitro RNA electrophoretic mobility shift assay (EMSA) with synthetic 42mer RNAs containing either m 6 A or A. The EMSA showed a direct binding between MhYTP2 and the m 6 A-modified MdMLO19 and MdMLO19-X1 mRNAs, with fourteen and eight "URUAY" motifs in their mRNA sequences, respectively (Appendix S1). (Figure 4b).
The result indicated that MhYTP2 binds to RNA transcripts that harbour m 6 A sites. Thus, MhYTP2, like human and Arabidopsis YTH-domain family proteins, was confirmed as an m 6 A reader protein. Therefore, the study's observations indicate that MhYTP2 binding with and degrading MdMLO19 and MdMLO19-X1 mRNAs may be a possible route via which MhYTP2 improved apple resistance to PM.

Overexpression of MhYTP2 promotes ribosome occupancy
Gene ontology (GO) analysis of the RIP-binding transcripts showed that the bound transcripts in 35S::MhYTP2 line OE-2 compared with the WT plants were enriched for plant-pathogen interaction and ribosome (Figure 4a). Recent studies showed that m 6 A modification regulates translation efficiency Zhou et al., 2021). Therefore, ribosome profiling was used to assess the ribosome density of each transcript mRNA with or without MhYTP2 overexpression to explore the role of MhYTP2 in mRNA translation. Overexpression of MhYTP2 changed the main distribution range of the translation efficiency data set (Figure 5a). Further analysis based on the abundance ratio of mRNA in the polysomal RNA versus the total RNA (ribosome occupancy) (Merchante et al., 2015) combined with RIP-seq results revealed that MhYTP2 overexpression enhanced the translation efficiency of MhYTP2 targets and non-targets (Figure 5b). GO analysis of the different ribosome occupancy transcripts (fold change ≥ 2; P < 0.05) showed that the transcripts were enriched in the antioxidation system (Figure 5c), suggesting the effect of MhYTP2 on the apple antioxidant system.

Overexpression of MhYTP2 enhances MdGDH1L translation efficiency
The different ribosome occupancy transcripts in 35S::MhYTP2 line OE-2 compared with the WT plants were enriched in the antioxidation enzymes (Figure 5c), including MdGDH1L, which affects the synthesis of GSH. The m 6 A-seq analysis of the 35S:: MhYTP2 line OE-2 and the WT plants showed that the MdGDH1L mRNA contains m 6 A sites at 3 0 UTR. The m 6 A modification level of MdGDH1L was significantly higher in 35S::MhYTP2 line OE-2 than that in WT plants (Figure 6a). The RNA immunoprecipitation-seq conducted using 35S:MhYTP2-Flag apple calli line MhYTP2-F2 ( Figure S2) showed that MdGDH1L mRNA was enriched in MhYTP2-flag IP mRNA samples (Figure 6b). Therefore, MhYTP2-His protein from BL21 Escherichia coli was expressed and purified to perform the in vitro RNA EMSA with synthetic 42-mer RNAs containing either m 6 A or A to gain insight into the MdGDH1L m 6 A binding activity of MhYTP2. The EMSA showed that MhYTP2 bound with m 6 A-modified MdGDH1L mRNA (Figure 6b). The MdGDH1L mRNA sequence contains seven "URUAY" motifs (Appendix S1). Furthermore, an overall increase in translation efficiency of the MdGDH1L mRNA was observed by ribosome  Figure 6c). Meanwhile, the activity of the GDH was significantly higher in 35S::MhYTP2 lines than in the WT (Figure 6d). Further evaluation revealed that GSH and AsA levels in 35S::MhYTP2 lines were higher than the WT plants under normal and PM inoculated conditions (Figure 6e). These findings indicate that MhYTP2 overexpression improved its target MdGDH1L mRNA translation efficiency leading to more MdGDH1L protein and elevated antioxidant activity, resulting in enhanced PM resistance (Figure 6f). These data suggest that critical gene in the antioxidation system undergo m 6 A-mediated post-transcriptional regulation, facilitating translation to increase protein content in response to stress.

MhYTP2 modulates genes encoding transcription elongation factors and translation initiation factor
Further, the effect of MhYTP2 on the translation efficiency of other genes involved in the antioxidation system was evaluated based on the abundance ratio of mRNA in the polysomal RNA versus the total RNA (Merchante et al., 2015). The genes Lascorbate peroxidase (APX) and peroxidase (PER) exhibited significant changes in translation efficiency when MhYTP2 was overexpressed (Figure 7a). Besides, the activities of APX and POD were significantly higher in 35S::MhYTP2 lines than in the WT ( Figure S3). This change could not be due to m 6 A deposition because the transcripts of these genes were not m 6 A-modified according to the m 6 A-seq data sets. Therefore, we speculated that MhYTP2 might regulate the translation efficiency of numerous transcripts beyond direct m 6 A modification. As expected, MhYTP2 overexpression altered the translation efficiency of mRNAs of several antioxidation-related genes, such as APX2, PER3, PER42 and PER47, which do not contain m 6 A modification.
Detailed analysis of the m 6 A-seq and RIP-seq data revealed that the transcripts of genes encoding transcription elongation factors (MD17G1038800 and MD14G1104300) and translation initiation factor (MD10G1126600) (Table S2), which play pivotal roles in facilitating protein synthesis by promoting the elongation of mRNA transcription and initiation of mRNA translation, respectively, exhibited m 6 A hypermethylation in the UTRs in 35S:: MhYTP2 line OE-2 (Figure 7b). RIP analysis showed direct interactions between MhYTP2 and the transcripts of MD17G1038800, MD14G1104300 and MD10G1126600 (Figure 7c). Taken together, MhYTP2 may modulate the translation efficiency of its non-targets indirectly by regulating m 6 A-modified mRNAs of transcription elongation factors and translation initiation factor in addition to modulation of translation efficiency via direct binding with the m 6 A-modified target, such as MdGDH1L mRNA. The results collectively indicate that MhYTP2 increased PM resistance by rapidly degrading MdMLO19 and MdMLO19-X1 mRNAs and increasing the antioxidant genes translation efficiency. Based on these results, a model has been proposed for the MhYTP2 function in apple PM disease resistance (Figure 8).

Discussion
Several researchers have investigated m 6 A of mammalians, and the discovery of m 6 A demethylases and the mapping of the m 6 A methylomes in the mammalian systems have indicated that m 6 A methylation of mRNA is a reversible and dynamic process with  . Studies have shown that YTHDF1 mediates translation and promotes translation efficiency  and YTHDF3 plays a significant role in modulating the translation of m 6 A-modified mRNAs (Li et al., 2017). Although the data available is scarce, the situation in plants seems to be similar. The Arabidopsis YTH-domain m 6 A reader protein ECT2  accelerated the degradation of three transcripts related to trichome morphogenesis, thereby affecting trichome branching (Scutenaire et al., 2018;Wei et al., 2018). Meanwhile, the present study focused on the m 6 A binding properties of MhYTP2 and its effect on mRNA and confirmed that the apple YTHdomain protein MhYTP2 is an m 6 A reader.
Overexpression of MhYTP2 in apple modified the expression levels of genes encoding m 6 A writers and erasers. The m 6 A writers with methyltransferase function for MdFIP37and that with demethylase function for MdALKBH6 were significantly downregulated in the 35S::MhYTP2 lines. Based on the finding that MhYTP2 modified the expression levels of genes encoding m 6 A writers and erasers, the transcriptome-wide m 6 A sequencing was conducted. Transcriptome-wide m 6 A distributions in the 35S:: MhYTP2 line OE-2 and WT apple plants indicated that the methylation sites are highly conserved compared with that in Arabidopsis and tomato (Scutenaire et al., 2018;Wei et al., 2018;Zhou et al., 2019), indicating a fundamental function of m 6 A in plants.
Further, the relationship between m 6 A distribution and gene expression was analysed. Importantly, the discovered features of the m 6 A distribution associated with gene expression in apple mRNAs were found different from Arabidopsis. The distinct enrichment of m 6 A in the exon regions was correlated with the overall upregulation of mRNA expression level in Arabidopsis (Scutenaire et al., 2018;Wei et al., 2018), while that in the apple was correlated with the overall downregulation of mRNA expression level in this study. Noticeably, m 6 A methylation at UTRs was positively correlated with gene expression. These observations indicated that the correlation between methylation level and gene expression depends on the modification site. Moreover, the m 6 A modifications in the 35S::MhYTP2 line OE-2 plants were highly enriched around the UTRs than that in the WT plants, which reveals that MhYTP2 may mainly affect the stability and translation of the m 6 A-modified mRNA (Anderson et al., 2018;Meyer et al., 2015). We speculate that the role MhYTP2 plays on the stability and translation of the m 6 A-modified mRNAs associated with disease resistance may affect apple defence against PM.
Studies have shown that the natural and artificial loss-offunction mutations of MLO S-genes reduce susceptibility to PM pathogens (Büschges et al., 1997). MdMLO19 induced by PM is a functional S-gene in apple, and its knockdown substantially reduced PM susceptibility (Pessina et al., 2016). Nevertheless, the regulatory mechanisms underlying the MdMLO19 signalling pathway, especially the regulation of the MdMLO19 gene at the post-transcriptional level, remain largely unknown. In this work, MdMLO19 and MdMLO19-X1, an isoform of MdMLO19, underwent m 6 A-mediated post-transcriptional regulation, which showed significant decrease in m 6 A modification levels of MdMLO19 in the 3 0 UTR and significant induction in m 6 A modification levels of MdMLO19-X1 in the exon regions, respectively, in the 35S::MhYTP2 line OE-2 compared with the WT plants. The changed m 6 A modification levels of MdMLO19 and MdMLO19-X1 mRNAs accelerated their degradation in 35S:: MhYTP2 line OE-2. These findings provide a novel information The m 6 A modification has also been demonstrated to affect translation efficiency beyond mRNA stability . The 35S::MhYTP2 line OE-2 of this study showed a global increase in m 6 A modification levels ( Figure 3a) and translation efficiency (Figure 5b). Upregulation of GDH genes has been observed under stress conditions (Fontaine et al., 2012;Lehmann and Skrok, 2010). In this study, the m 6 A modification at 3 0 UTR and translation efficiency of MhYTP2-binding MdGDH1L were significantly higher in the 35S::MhYTP2 line OE-2 than in the WT plants (Figures 6a, c). Further, the changes in translation efficiency appeared to be directly regulated by m 6 A deposition on the transcripts of MdGDH1L or indirectly by m 6 A-mediated regulation of transcription elongation factors and translation initiation factor for MdAPX2, MdPER3, MdPER42, and MdPER47. The improved translation efficiency of MdGDH1L made the transgenic plants resistant to PM. These findings collectively reveal a novel layer of gene regulation in the antioxidation system signalling pathway and establish a link between the m 6 Amediated antioxidation system and apple disease resistance.
The present study identifying the function of the apple m 6 A reader MhYTP2 in mRNA stability and translation sheds light on the mechanisms through which m 6 A functions in RNA metabolism and plant disease resistance. Considering the multiple roles of MhYTP2 on m 6 A metabolism, the regulation identified may have an essential function in many biological processes.

WT and 35S::MhYTP2 plant treatment
The WT plants and the 35S::MhYTP2 plants of the Malus domestica cv. 'Roya Gala' were used in this study . The WT and 35S::MhYTP2 plants generated by tissue culture were initially grown on MS agar containing 0.2 mg/L 6benzylaminopurine (6-BA) and 0.2 mg/L indoleacetic acid (IAA) at 23°C, 60 µmol/m 2 /s, and 14 h photoperiod. After 45 days of growth on MS agar, the plantlets were transferred to the rooting MS agar media containing 0.5 mg/L indole butyric acid and 0.5 mg/L IAA and maintained for another 45 days. The WT and 35S::MhYTP2 plantlets with similar size were transferred to small plastic pots (8.5 cm × 8.5 cm × 7.5 cm) containing a mixture of soil and perlite (1 : 1, v : v). After 60 days of growth in a growth chamber under 50% relative humidity, 28°C, and a long photoperiod (16 h:8 h, light:dark), plants of similar size were selected to test the response to PM inoculation.
The inoculation experiments following the dry-brushing of healthy leaves with P. leucotricha obtained from the diseased leaves were carried out in a growth chamber at 50% relative humidity and 28°C in the dark for 24 h. Disease severity on all inoculated leaves was visually assessed 15 days after the inoculation. Six leaves per plant were harvested before and after inoculation and immediately frozen in liquid nitrogen. All frozen samples were stored at −80°C. The phenotype recording and trypan blue and leaf fungal hyphae WGA staining were carried out 15 days after inoculation. In addition, the disease severity under field conditions was determined manually based on the size of white spots on leaves.
Microscopy WGA-AF 488 (Molecular Probes, Karlsruhe, Germany) was used to stain the fungal hyphae, and propidium iodide (Sigma, Shanghai, China) to stain the plant cells. Samples were infiltrated with a staining solution (1 µg/L propidium iodide, 10 µg/L WGA-AF 488; 0.02% Tween 20 in PBS pH 7.4), incubated for 30 min, and washed in 1× PBS (137 mM NaCl, 2.7 mMKCl, 12 mM phosphate buffer, pH 7.4). Images were recorded on a TCS-SP5 confocal microscope (Leica, Bensheim, Germany), at an excitation wavelength of 488 nm and a detection wavelength of 500-540 nm. The mCherry fluorescence (an excitation wavelength of 561 nm and a detection wavelength of 580-630 nm) was used for live-cell imaging of fungal hyphae in apple leaves. Green-fluorescence protein was excited with a 488 nm laser, and the emission was detected at 495-530 nm for fungal hyphae. The samples were observed, and images were recorded using a CoolSNAP-HQ chargecoupled device camera (Photometrics, Shanghai, China) controlled with the imaging software MetaMorph (Universal Imaging, Los Angeles, CA, USA).

GDH activity assay and antioxidant metabolite analysis
GDH activity and the GSH and AsA content of apple leaves were determined using the specific kits (Sangon Biotech, Shanghai, China), following the manufacturer's instructions.

RNA extraction and quantification and gene expression analysis
Total RNA was extracted from the collected samples using a spin column plant total RNA purification kit (Sangon Biotech), according to the manufacturer's instructions. The cDNA was reverse transcribed from the total RNA using a PrimeScript ® RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Further, quantitative RT-PCR was conducted on a LightCycler ® (Roche, Basel, Switzerland) 96 real-time PCR detection system (Roche) using the ChamQ SYBR quantitative PCR Master Mix (Vazyme Biotech, Nanjing, China), according to the manufacturer's instructions. The apple MdActin (XM_008344381) was used as the internal control gene. The relative expression level of the target gene transcript was determined by the 2 ÀΔΔCt method (Kim et al., 2009;Livak and Schmittgen, 2001). All primers are listed in Table S3.

RIP-seq and EMSA
RNA immunoprecipitation was performed as previously described by Zhou et al. (2019). The 35S::MhYTP2-Flag 'Orin' cultivar's calli were flash-frozen and crushed in liquid nitrogen, and the frozen powder was homogenized in 15.75 mL of lysis buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% IGEPAL, and 1% plant protease inhibitor cocktail from Sigma). The crude extract was clarified by centrifugation at 3000 g for 10 min at 4°C. The immunoprecipitation was performed using 11.25 mL of the crude extract incubated for 1.5 h at 4°C on a rotating wheel with 120 µL of Flag-trap magnetic beads (Chromotek, Munich, Germany). RNA was eluted from the beads with 400 µL of guanidium extraction buffer and precipitated overnight at −22°C with 800 µL of pure ethanol. RNA was precipitated by centrifugation, resuspended in 350 µL of RTL buffer (Qiagen, Hilden, Germany), purified according to the manufacturer's instructions, and eluted in 15 µL of RNase-free water. The RNA solution was concentrated to 6 µL using the RNA clean and concentrator kit (Zymo Research, Irvine, CA, USA. Code: R1015). Similarly, RNA was extracted from 200 µL of crude extract to monitor RNA in the input fraction. RNA samples from both the eluate and input fractions were treated with DNase as part of the Qiagen RNEasy purification procedure. The beads were resuspended in 15 µL of Laemmli buffer and incubated for 5 min at 95°C, and the supernatant was collected from the beads using a magnetic device. Sequencing was done on an Illumina HiSeq machine with 2 × 100 cycles of Solexa pairedend sequencing. The experiment was repeated three times. Whole RIP and protein immunoprecipitation experiments were conducted using the freshly prepared and harvested calli for each replicate.
Electrophoretic mobility shift assay was performed following a previously reported method (Wang et al., 2014;Wei et al., 2018) with minor modifications. The digoxin-labelled RNA oligonucleotides used to assay the binding affinity of MhYTP2-His are listed in Appendix S2. The RNA probe was used at 4 nmol concentration. The concentration of MhYTP2-His ranged from 0 to 2000 nM.

mRNA purification and m 6 A-seq
Total RNA was extracted from the collected samples using a spin column plant total RNA purification kit (Sangon Biotech). Intact mRNA was purified from total RNA using Dynabeads mRNA purification kit (Ambion, Austin, TX, USA. Code: 61006).
The m 6 A IP was carried out following a previously reported procedure with slight modifications (Chen et al., 2015;Dominissini et al., 2013). The purified mRNA samples from the WT and 35S::MhYTP2 line OE-2 plants were digested using DNase I to generate 100 nucleotide-long fragments by incubating at 94°C for 5 min in RNA fragmentation buffer (10 mM ZnCl2, 10 mM, Tris-HCl, pH 7.0). The reaction was stopped using 0.05 M EDTA (Ambion Code: AM8740), followed by phenol-chloroform extraction and ethanol precipitation. For the m 6 A-seq, anti-m 6 A polyclonal antibody (10 µg antibody for 5 µg mRNAs; Synaptic Systems (Chromotek. Code: 202003)) was incubated in IP buffer containing 150 mM NaCl, 0.1% NP-40 (v/v), 10 mM Tris-HCl (pH 7.4), and 300 U/mL RNase inhibitor (Promega, Madison, WI, USA. Code: N2112S) for 2 h at 4°C. The mixture was IP by incubating with 50 µL Protein A beads (Sigma Code: P9424) at 4°C for another 2 h. After washing twice with high-salt buffer consisting of 50 mM Tris-HCl (pH 7.4), 1 M NaCl, 1 mM EDTA, 1% NP-40 (v/v), and 0.1% SDS (w/v) and twice with IP buffer, bound mRNAs were eluted from the beads by incubating with 6.7 mM N 6methyladenosine (Sigma Code: M2780) in IP buffer and recovered via phenol-chloroform extraction and ethanol precipitation. Then, 50 ng of the IP mRNA or pre-IP mRNA (input control) was used for library construction with NEBNext ultra RNA library prep kit for Illumina (NEB, Beijing, China. Code: E7530). High-throughput sequencing was performed on the Illumina HiSeq X sequencer with a paired-end read length of 150 bp according to the standard protocol (Zhou et al., 2021). The sequencing was carried out with two independent biological replicates, and each RNA sample was prepared from a mix of at least 10 apple leaves.

RNA-seq
The input reads of the m 6 A-seq were used for RNA-seq analysis as previously described (Trapnell et al., 2010). The uniquely mapped reads of each sample were assembled by Cufflinks. Gene expression level was calculated as fragments per kilobase of exon per million mapped fragments (FPKM) using Cuffdiff, which provides statistical routines for assessing the differential gene expression levels (Trapnell et al., 2010). Differentially expressed genes were defined based on a cut-off criterion of FPKM fold change ≥2 and P < 0.05.

GO and KEGG analyses
The clean reads obtained after removing the adapter and lowquality reads were aligned to the apple reference genome GDDH13 by HISAT2 (Kim and Langmead, 2015). The fea-tureCounts (Liao and Smyth, 2014) was used to count the reads mapped to each gene. The DEGs of Ribo-seq and RIP-seq were further subjected to GO analysis on the agriGO database (version 2.0; http://systemsbiology.cau.edu.cn/agriGOv2/). Meanwhile, the DEGs of m 6 A-seq were subjected to KEGG (http://www. genome.jp/kegg/) analysis.

mRNA stability assay
The mRNA stability was assessed as previously described (Duan et al., 2017) with minor modification. Briefly, tissue-cultured WT and 35S::MhYTP2 line OE-2 plants were grown on modified MS agar with 0.2 mM actinomycin D (transcription inhibitor), as described in the section on WT and 35S::MhYTP2 plant treatment. The tissues were collected 0, 5, 15 and 20 days after adding transcription inhibitor and immediately frozen in liquid nitrogen. The tissues were stored at −80°C until further gene expression analysis.

Western blot analysis
The proteins were separated by 10% SDS-PAGE and transferred to an Immobilon-P PVDF membrane for western blotting. The membrane was blocked with 5% non-fat milk in PBST buffer for 2 h at room temperature. The immunoblotting was conducted by incubating with anti-GDH1L antibody (1 : 10 000) at room temperature for 2 h, followed by incubation with HRP-conjugated antirabbit IgG secondary antibody (1 : 10 000) at room temperature for another 2 h. The immunoreactive bands were visualized using the enhanced chemiluminescence detection kit, as mentioned earlier. The antibodies were synthesized from the Wuhan Institute of Biotechnology (China) (www.atagenix.com).

Statistical analysis
All data were analysed using IBM SPSS Statistics 21 (IBM Corp., Chicago, IL) and graphed with Sigma Plot 12.0 software (Systat Software, CA). Data are presented as the mean AE standard error (SE) of the mean of triplicates for each measurement. Data were analysed using an independent t test (P < 0.05) or subjected to one-way analysis of variance (ANOVA).

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.
Appendix S1 m 6 A domain analysis of mRNA sequences. Appendix S2 Digoxin labelled RNA oligonucleotides of MdMLO19, MdMLO19-X1 and MdGDH1L for EMSA. Figure S1 MhYTP2 conserved motifs analysis of YTH-domain family proteins in Arabidopsis and human. Figure S2 Confirmation of transgenic 35S::MhYTP2-Flag apple callus lines MhYTP2-F1 and MhYTP2-F2. Figure S3 Changes in APX and POD enzyme activities in apple leaves under normal and infected conditions. Table S1 The amino acid sequence alignment of MdMLO19-X1 identified by us and MdMLO family reported in the literature (Pessina et al., 2014).